Project Summary/Abstract Streptococcus agalactiae (group B Streptococcus; GBS) is the most common infectious cause of neonatal morbidity and mortality in the United States and a major global contributor to stillbirth and infection in the newborn period. While asymptomatic genitorectal GBS colonization is common among healthy adults, vaginal colonization in late pregnancy is a major risk factor for neonatal infection. For this reason, improved understanding of bacterial genes that promote vaginal colonization and the transition from a colonizing to an invasive lifestyle may open avenues to new preventative strategies. Our group has constructed highly saturated GBS transposon mutant libraries, which have been instrumental in genome-wide assessments of gene contributions to bacterial fitness in a variety of experimental settings. Recently, we developed a high- throughput strategy for using these complex, intermixed libraries to generate comprehensive, ordered mutant libraries of nonessential GBS genes (of which there are approximately 1,700). In Aim 1, we propose to use techniques and technologies we have developed in order to assemble complete, indexed mutant libraries of two GBS strains, representing common capsular subtypes recovered from infected patients (serotypes Ia and V). These indexed libraries?which will be freely shared with the research community at large?will represent a major advance in pathogenesis research, allowing study of single mutants or curated library sub-pools in a wide range of experimental designs. In Aim 2, we will use the indexed libraries in a series of studies to fully characterize the contribution of GBS two-component signaling pathways to vaginal colonization in a mouse model. Library sub-pools containing knockouts of all 19 GBS two-component systems will be assessed for constituent mutants with significant fitness defects in the vaginal colonization model. Top candidates will be validated with targeted isogenic knockout strains, which will also be characterized via RNA-seq to identify downstream genes regulated by each candidate two-component system. Finally, indexed library knockouts of each candidate two-component system regulon will be assessed for major fitness contributors in the vaginal colonization model. These aims will establish new, flexible, and powerful tools for studying GBS pathogenesis. They will permit unbiased and comprehensive assessment of two-component systems and their regulated genetic networks as potential targets for preventing neonatal infection.